From www.bloodjournal.org by guest on December 22, 2014. For personal use only. Differentiation-Associated Changes in CD44 Isoform Expression During Normal Hematopoiesis and Their Alteration in Chronic Myeloid Leukemia By S. Ghaffari, G.J. Dougherty, P.M. Lansdorp, A.C. Eaves, and C.J. Eaves CD44 is a widely expressed, multifunctional, cell-surface glycoprotein thathas been implicated in the regulationof normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique t o CD44 isoforms containing amino acids encoded by thealternatively spliced exon v10, t o compare the expression of CD44 on primitive hematopoietic cells from the marrowof normal individuals and theirneoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameterfluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed thatCD44 is normally expressed at high t o very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors(burst-forming units-erythroid [BFU-El) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed thevery high levels of CD44 seen on 20% to 2540 of LTC-IC and CFU-GM. Antibody staining showed the expression of exon vl0-containing CD44 isoforms t o be restricted t o a small subpopulation(490t o 896)of morphologically recognizable mature (CD34-) myeloid cells within the light-density fractionof normal marrowcells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon vl0-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplasticCFU-GM expressed very high levels of CD44. and these CFU-GM were accompanied by an increased number oflight density v10' cells, including some that coexpressed CD34. Nonmalignanthematopoieticprogenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative t o normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normalhematopoiesis in vivo that may be deregulatedin theneoplastic clone of patients with CML. 0 1995 by The American Society of Hematology. I homing of primitive hematopoietic cells into the Neoplastic transformation may also alter the adhesive characteristics of primitive hematopoietic cells with associated changes in their turnover and tissue distribution. For example, in chronic myeloid leukemia (CML), a multilineage clonal malignancy believed to arise as a result of the formation of a BCR-ABL fusion gene in a pluripotent hematopoietic stem cell,' the leukemic progenitors exhibit abnormal adhesive properties, are found at abnormally elevated levels in the blood, and are able to establish hematopoiesis in extramedullary sites.'." CD44 is an adhesion molecule that is expressed on the surface of many cells, including representatives of all hematopoietic cell lineages."-16 Hyaluronan is the mostwidely recognized ligand of CD44, but evidence of binding to fibronectin, collagen, and serglycin has also been rep~rted.''.'~ The common form of CD44 expressed on hematopoietic cells (CD44H) isan 85- to 90-kD glycoprotein. A large number of higher-molecular-weight isoforms may also be produced in specific cell types or under specific conditions as a result of the alternative splicing of at least 10 contiguous exons (v1 through v10) within the CD44 gene.'"'" CD44R1 is one of several vl0-containing CD44 cDNAs. It was cloned from the KG- 1 a leukemic cell line and contains in the extracellular region of the molecule an insertion of 132 amino acids encoded by exons v8, v9,and v10." This isoform corresponds to domains IVand V of the original CD44 clones and to the epithelial form of CD44 (CD44E), from which it differs by only three amino acids." CD44R2 (also referred to as domain V) is avl0-containing isoform of CD44 that shares only the last 69 amino acids present in the unique region of CD44R1. Both the expression and binding properties of CD44 on normal cells appear to be subject to regulation.'6 In addition, changes in CD44 isoform expres- N ADULT LIFE, THE proliferation and differentiation of primitive hematopoietic progenitors is normally restricted to the bone marrow, where these cells interact with various stromal cells of nonhematopoietic origin, aswell as with a complex mixture of extracellular matrix (ECM) components. Such interactions are believed to regulate the accessibility of these cells to stimuli that control their viability, cell-cycle progression, and movement into and out of the circulation. These concepts are based in part on the identification on the surface of primitive hematopoietic cells of cytokine receptors and adhesion molecules with affinities for various membrane or ECM-bound ligands.',' In addition, in vivo experiments have shown that particular ligands, or antibodies for specific cell adhesion molecules, may stimulate the rapid exodus of primitive hematopoietic cells from themarrow into the blood or, conversely, may alter the From the Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver; and the Departments of Pathology and Laboratory Medicine, Medicine,and Medical Genetics, University of British Columbia,Vancouver, Canada. Submitted March 27, 1995; accepted June 14, 199.5. Supported by the National Cancer Institute of Canada (NClCJ and the Canadian CancerSociety. S.G. is a recipient of u Terry Fox Physician-Scientist Fellowship of the NClC. G.J.D. is a Research Scientist of the NCIC, and C.J.E. is a Terr). Fax Cancer Research Scientist of the NCIC. Address reprint requests to C.J. Eaves, PhD, Terty Fox Laboratory, 601 W 10th Ave, Vancouver, BC VSZ IL3, Canada. The pubiicationcosts of this article were defrayed in partby page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8608-0020$3.00/0 2976 Blood, Vol 86, No 8 (October 15). 1995: pp 2976-2985 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CD44EXPRESSIONONNORMAL 2977 AND CML CELLS sion have been found to characterize various metastasizing cells, such as those present in certain aggressive lymphomas23-25 and in a variety of transformed epithelial cell populat i o n ~ . *Evidence ~ - ~ ~ that CD44 may be involved in the regulation of early stages of normal hematopoiesis has been suggested by the observations that primitive clonogenic cells express CD44I5 and that the addition of anti-CD44 monoclonal antibodies to long-term marrow cultures (LTC) can cause a marked and sustained decrease in the number of mature cells subsequently found in the nonadherent fraction of these ~ u l t u r e s . Whether ~ ~ ~ ~ ' this is due to an effect on the primitive hematopoietic cells that normally become part of the adherent cell layer in this culture system has not been established. Indeed, the level of expression of CD44 on cells that give rise to clonogenic progenitors under these conditions in vitro has not been previously characterized. Similarly, in spite of the well-known abnormal adhesive properties of CML cells, potential alterations in their expression of CD44 have not been investigated. In this study, we show that there are quantitative differences in the levels of CD44 expressed on primitive normal erythroid (burst-forming unit-erythroid [BFU-E]) and granulopoietic(colony-forming unit-granulocyte/macrophage [CFU-GM]) progenitors and on their precursors identified functionally as LTC-initiating cells (LTC-IC). We have also identified a small subset of CD34- cells in normal marrow that express CD44 isoform(s) containing the amino acids encoded by the alternatively spliced exon v10. Interestingly, quantitative changes in both of these aspects of CD44 expression were found when predominantly leukemic populations isolated from patients with CML were studied. These changes include an increase in the proportion of CFU-GM expressing very high levels of CD44 and the expression of the exon vl0-containing CD44 isoform(s) on CD34' neoplastic cells. MATERIALS AND METHODS Cells. All samples of human peripheral blood (PB) and bone marrow (BM) were obtained from informed and consenting individuals. Samples of normal BM were from harvests taken for allogeneic transplantation or from normal cadaveric tissue. Some samples were also obtained from leukapheresis harvests collected from patients with hematologic diseases in remission (two with Hodgkin's disease and one acute myeloid leukemia [AML]). The two patients with Hodgkin's disease received chemotherapy (cyclophosphamide, 7 g/ m') followed by administration of interleukin-3 (IL-3; 2.5 to 5 pgi' kg/d) and granulocyte-macrophage colony-stimulating factor (GMC S F 5 pgkgld) until the leukaphereses were completed. The patient with AML received G-CSF (12 pg/kg/d) for 5 days followed by 2 days of leukapheresis. Heparinized PB samples were obtained from patients with newly diagnosed Philadelphia chromosome-positive (Ph') chronic phase CML undergoing routine hematologic assessment (Table 1). In all but one case, the CML cells had been cryopreserved in 90% fetal calf serum (FCS; StemCell Technologies, Vancouver, Canada) and 10% dimethyl sulfoxide and then stored at -70В°C before use. To ensure that the results of analyses of CML samples could be attributed to leukemic progenitor populations, only PB samples that were found to contain clonogenic cells that were present at frequencies at least 25-fold above themean for fresh normal blood were used (Table 1). All analyses were performed on Table 1. Initial WBC and Clonogenie Progenitor Concentrations in Fresh or Cryopresewed PB Samples From Patients With CML Sample No. WBC Count (xlOe/mLi la* 1 b' 180 180 2 250 3 450 350 300 4 5 Status When Used F T T T T T BFU-EtmL 7,200 7,400 CFU-GMImL 2,700 2,200 400,000 150,OOO 150,000 970,000 780,000 380,000 190,000 120,000 Abbreviations: F. fresh; T, subsequently thawed. * Progenitor values for samples la and l b are from the same patient. cells in the light-density fraction (less than 1.077 g/mL) isolated using Ficoll-hypaque (Pharmacia LKB, Uppsala, Sweden). Antibodies. lgGl monoclonal antibodies (MoAbs) specific for CD34 (8G12),32Thy-l (5E10),33and an epitope located on the common NH,-terminal region of different isoforms of CD44 (3C12))4 were purified from tissue culture supernatants using Protein G chromatography (Pharmacia LKB). MoAb 8G12 labeled with Cyanine5 (Cy5) has been described previously32and was used at 10 pg/mL. MoAb 5E10 was labeled with phycoerythrin (PE; Pharmingen, San Diego, CA) and used at 5 &mL, and 3C12 was used at 1 pg/mL. MoAb 2G1 is an IgGl MoAb that reacts with an epitope encoded by exon ~ 1 0 . (In ' ~ this study, cells expressing exon vl0-containing CD44 isoforms are referred to as 2G1+ cells or vl0' cells.) MoAb 2G1 was used as a hybridoma tissue culture supernatant. An irrelevant IgGl MoAb (anti-de~tran)~~ was used as a control in all staining and sorting experiments. A goat F(ab'), anti-mouse IgG (H + L) preparation labeled with fluorescein isothiocyanate (GAM-FITC) was purchased from Caltag Laboratories (South San Francisco, CA). Staining andfrow cytometry. Cells were washed twice and resuspended in Hank's balanced salt solution with 2% FCS and 0.02% sodium azide (NaN,) (HFN). All staining procedures were indirect and were performed with the cells at a concentration of107/mL. Cells were first incubated with HFN containing 5% human serum (HS) at room temperature to block Fc receptors, then washed twice with HFN, and incubated with an anti-CD44 MoAb (3C12, 2G1) or the IgGl control MoAb for 30 minutes at 4"C, followed by two washes with HFN. Samples were then resuspended in a 150 dilution of GAM-FITC in HFN, incubated for 30 minutes at 4"C, washed twice, and incubated for another 30 minutes at 4В°C with 200 pg/mL of the irrelevant mouse IgG MoAb to block residual GAM-FITC. (The use of a high concentration of this antibody was necessary to block the residual GAM-FITC on the surface of cells expressing high numbers of CD44 molecules.) The anti-CD34 MoAb (8G12Cy51 andanti-Thy-l MoAb (5ElO-PE) were then added to this solution without further washing in the presence of the irrelevant mouse IgG for another 30 minutes at 4В°C. Cells were then washed twice with HFN, with the second wash containing propidium iodide (PI; Sigma Chemicals, St Louis, MO) at 2 wg/mL to stain dead cells. Sorting of cells was performed on a FACStar Plus (Becron Dickinson, San Jose, CA) equipped with a 5-W argon and a 30-mW helium neon laser (Spectra-physics, Mountain View, CA).In all experiments, gates were set to exclude dead cells (PI+) as well as most erythrocytes and granulocytes as defined by their forward light scatter (FSC) and side scatter (SSC) characteristics. Sorted cells were collected in Iscove's Dulbecco's modified Eagle's medium (DMEM) (STI) containing 2% FCS and were kept on ice until plated. Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The following PCR primers corresponding to CD44 se- From www.bloodjournal.org by guest on December 22, 2014. For personal use only. GHAFFARI ET AL 2978 A B .. - .:. I n FSC FSC D C d. c') n 0 ssc quences located in the 5' common region and in the 3' alternatively spliced exon v10 were used: 5'(5'-TGTACATCAGTCACAGACCT-3') and ~'(S-AGGAACGATTCIACATTAGAG-~').Total RNA was isolated using TRIzol Reagent (Gibco BRL, Gaithersburg, MD). The first cDNA strand was generated in a 50-pL reaction containing 5 pg of total RNA, 0.5 mmol/L deoxynucleotide triphosphates (dNTPs), 200 pm01of random hexamers, 4 mmol/L dithiothreitol, 200 U of superscript reverse transcriptase (Gibco BRL), and 2.5 U of human placental RNase inhibitor (Gibco BRL). The reaction was incubated at 42В°C for 1 hour followed by 5 minutes at 95В°C to inactivate the enzyme. Then I O pL of this reaction was subjected to PCR in a 100-pLvolume of 25 mmol/L KCI, 1.5 mmol/L MgC12, 0.2 mmol/L dNTPs, 1 0 0 pmolof each primer, and2.5 U ofTaq polymerase, Thirty cycles were performed as follows: 30 seconds of denaturation at 94В°C. 30 seconds of annealing at 48В°C.and 3 minutes of extension at 72В°C. This was followed by a final extension at 72В°C for 5 minutes. The PCR products were separated on a 2% agarose gel, transferred to a nylon membrane, and hybridized to a 196-bp vl0-specific probe obtained by PCR of the CD44R1 cDNA2' using the primers 5'(5'-TAGGAATGATGTCACAGGTG-3')and 3'(5'-AGGAACGATTGACATTAGAG-3'). Hybridization was performed overnight at 60В°C in 6X saline sodium citrate (SSC; 1X SSC is 0. I5 mol/L NaCI, 0.01 5 mol/L sodium citrate, pH 7.5). 1% sodium dodecyl sulfate (SDS), 0.02% polyvinylpyrrolidone, 0.02%ficoll, 0.02% bovine serum albumin (BSA), 10 pg/mL of denatured salmon sperm DNA, and 10' c p d m l of denatured probe. The filter was then washed at a final concentration of 0.1 X SSC, 1% SDS at 65В°C. Autoradiography was performed at room temperature withKodak XAR-5 film for 14 hours (Eastman-Kodak, Rochester, NY). CD44 Fig 1. Gates used for analysis of antibody reactivity with normalBM cells. BM cells were gated to exclude dead cells (A) and cellswith high SSC and very low FSC (B). The gating used to select cells with high CD34expressionis shown in panel C. The gating used to subdivide the CD34'cellsaccording to their expression of CD44 and Thy-l is shown in panel D. The four quadrants shown in panel D represent the fourCD34subpopulations analyzed functionally: CD44++Thy-l-, CD44"Thyl*, CD44"*Thy-l-. and CD44"'Thy-l'. The same gates were used for CML PB and mobilized blood. Hematopoietic progenitor assays. Cells from primary samples or LTC harvests were assayed for clonogenic erythropoietic (BFUE), granulopoietic (CFU-GM), and multilineage (CFU-granulocyte, erythroid, monocyte, megakaryocyte: CFU-GEMM) progenitors in Iscove's DMEM-based methylcellulose cultures containing 3 UlmL of human erythropoietin; 20 n g h L each of granulocyte colonystimulating factor (G-CSF; Amgen, Thousand Oaks, CA), granulocyte-macrophage CSF (GM-CSF; Sandoz, Basel, Switzerland), IL3 (Sandoz), and IL-6 (obtained from the supernatant of COS cells transfected with a full copy human IL-6 cDNA; D. Hogge, Terry Fox Laboratory, Vancouver, Canada); and 50 ng/mL of Steel factor (SF; Amgen). The methodology and criteria for hematopoietic colony generation and recognition were the same as previously de~cribed.~' The general procedure used for LTC-IC assays has also been described in detail previously." Briefly, test cells were resuspended in long-term medium (an enriched Alpha medium containing 12.5% horse serum, 12.5% FCS, mol/L 2-mercaptoethanol [STI], to which freshly dissolved hydrocortisone sodium hemisuccinate [Sigma] was added just before use to give a final concentration of moVL) and then seeded onto semiconfluent feeder layers of irradiated (80 Gy) mouse marrow-derived fibroblasts that had been genetically engineered to produce human G-CSF, L-3, and SF.3q These LTC-IC assay cultures were then maintained at 37В°C and fed weekly by replacement of half of the medium containing half of the nonadherent cells with the same volume of fresh long-term medium. After a total of 6 weeks, the nonadherent cells were removed, washed, and combined with the trypsinized and suspended adherent layer cells. These pooled cells were then plated in methylcellulose assays as described above. The total number of clonogenic cells From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CD44 EXPRESSIONON 2979 NORMAL AND CML CELLS 103 104 CD44 Fig 2. Expression of CD44 versus Thy-l on CML PBCD34* cells located within t h e gates shown in Fig 1. (BFU-E plus CFU-GM plus CFU-GEMM) present at 6 weeks provides a relative measure of the number of LTC-IC originally present in the test suspension." Limiting dilution experiments have shown that under the conditions used herefor LTC-IC detection, on average, one LTC-IC will produce approximately eight clonogenic cells (C.J.E., unpublished observation, August 1995). and this was, therefore, the value used to derive the absolute LTC-IC frequencies reported. In LTC-IC assays where no colonies were detected, the minimum number of LTC-ICthat could have been detected was calculated by assuming that one colony had been produced by the entire aliquot of cells evaluated in the final methylcellulose assays. This value (instead of zero) was then used to derive an estimate of the upper limit of the mean 5 SEM number of LTC-IC in groups where one or more values were below the limit of detection. In such cases the mean is indicated as less than x 5 SEM. RESULTS Altered CD44 expression on phenorypically dejined populations of primitive CML cells. To compare the patterns of expression of CD44, CD34, and Thy-l on primitive normal and neoplastic (Ph') CML cells, we isolated various fractions of light-density bone marrow cells from normal individuals and compared their staining profiles with those obtained for cells in the light-density fraction of PB from a series of patients with newly diagnosed CML and high white blood cell (WBC) counts. The choice of this source of CML cells for these comparisons was based on previous data showing that the light-density cells in the PB of patients with chronic phase CML and high WBC counts, on average, will contain a greater than 10-fold increase in all types of leukemic (Ph') progenitors (both clonogenic cells and LTCIC), such that they approach or even exceed the frequency of the same types of primitive normal cells in the lightdensity fraction of normal BM cell^.^^" Figure 1 shows representative scatter plots for a normal BM sample, and Fig 2 shows analogous plots for a representative CML PB sample stained in the same way. These figures illustrate the general finding for all samples studied: 290%of the light-density cells in both normal BM and CML PB were CD44'. In addition, among the CD34' populations cells were detectable. present in these samples, no CD" From these analyses, two additional similarities between normal BM and CML PB were consistently noted. First, the relative proportion of CD34' cells that were also Thy-l' was approximately the same. Second, the proportion of (total) CD34' cells in CML PB expressing very high (1,OOOX as compared with the background, designated CD44"'), proportion expressing intermediate (IOOX background, CD44++),levels of CD44 appeared indistinguishable from those characteristic of the CD34' population in normal BM (Table 2). However, in the CML samples, the proportion of Thy-]' cells that were CD44"' was significantly higher than in normal BM (0.4% t 0.1% of CD34' cells in normal BM compared with 1.2%? 0.2%of CD34' cells in CML PB; P < .01, Student's t test). In addition, we looked within the entire light-density fraction of normal BM and CML PB, as well as within their respective CD34' subpopulations, for cells expressing exon vl0-containing CD44 isoforms. For this, we used the 2G1 MoAb, which specifically recognizes an epitope present on Table 2. Comparisons of the Distribution of Total CD34* Cells and the Total Light-Density Cells by Their Levels of Expression of CD44 and Thy-l in Normal BM Versus CML PB CD44" Population Evaluated and Origin of Cells Total LDF Normal BM CML PB CD44" * no Thy-l- Thy-l* 6 351 321 80 e 1 77 e 3 82 e 1 80 2 1 0.4 2 0.1 1.2 t 0.2 17 2 1 18 t 3 0.2 z 0.1 0.4 ? 0.1 4t2 11 e 8 422 11 2 8 0.05 2 0.02 0.3 t 0.02 0.9 t 0.3 423 6 Total Thy-l- Thy-l' Total 18 t 1 19 2 1 CD34' Normal BM 6 CML PB 6 1 2 0.3 4e3 Values shown are the mean 2 SEM of the individual values for the various populations evaluated, expressed in each case as a percent of the total LDF. Abbreviation: LDF, light-density fraction. Number of samples, for six normalindividuals and five patients with CML. One of the samples from the first patient with CML was studied twice (before and after thawing; see Table 1). From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 2980 GHAFFARI B A I CD44 common region Alternatively spliced I v2 Extracellular -D Domain I v3 Cytoplasmic Domain I I v4 I 680 bp I v5 480 bp sequences 4- - W Fig 3. RT-PCR analysis of 2G1+ cells. (A) Primers (arrows) were designed t o amplify all vl0-containing CD44 isoforms and t o exclude all isoforms lacking thisexon. The shaded area corresponds t o the transmembrane domain. Human v1 contains an in-frame stop codon. (B) PCR products were separated on a 2% agarose gel, transferred t o a nylon membrane, and hybridized t o a v10-specific probe (see Materials and Methods). The arrows indicate bands corresponding t o expected reaction products forCD44R1 and CD44R2. the extracellular portion of CD44 isoforms containing the amino acids encoded by the v10 exon. In normal BM, 2G1+ cells were relatively rare, comprising from 4% to 8% of the total light-density fraction and s l % of the CD34' cells. In all of the six CML PB samples analyzed, 2GI' cells were much more prevalent (up to 30% of the light-density cell fraction) and included cells that were also expressing CD34 (up to 7%), although there was marked patient-to-patient variability in the proportion of CD34' cells that were also 2G l +.Nevertheless, because of the large elevations in total numbers of light-density myeloid cells in the blood of these patients, these findings suggest that there is a marked in- crease in the absolute number of 2GI' hematopoietic cells in patients with CML. On the other hand, in both normal BM and CML PB, allof the 2G1' cells appearedtobe Thy- 1 -. Fluorescence-activated cell sorter (FACS) analysis further showed that the light-density 2G 1 cells from either normal BM or CML PB had lowSSC properties and intermediate to highFSC characteristics. After sorting andMayGrunwald Giemsa staining, all 2G1' cells, regardless of their origin, appeared to be exclusively myeloid cells (and did not include erythroblasts or other recognizable cell types). In functional assays of the sorted CD34' 2G1+ CML cells, no clonogenic progenitors of any kind were detected. + Table 3. Progenitor Distributions in Subpopulations of CD34' Cells in Normal BM by Their Levels of Expression of CD44 and T h y l No. of Progenitors/105 Cells' CD44"' CD44" Tvoe of Progenitor Evaluated Sample No. BFU-E 2 3 4 5 6 Mean ? SEM CFU-GM 2 3 4 5 6 Mean 2 SEM LTC-IC* 2 3 4 5 6 Mean t SEM Thy-l + 5,000 ND 1,300 2,100 2,500 ND 2,700 2 800 3,800 ND 23,000 9,000 7,700 ND 11,000 2 4,200 ND 18,000 1,800 1,600 3,300 2,900 5,600 2 3,200 Thy-l- 7,500 8,000 5,000 5,700 13,000 9,700 8,100 2 1,200 15,000 13,000 19,000 6,700 6,200 9,500 11,000 2 2,000 ND 900 80 200 900 800 600 ? 200 Thy-l- Thy-l' ND ND < 1,000 ND ND ND < 1,000 ND ND 15,000 ND ND ND ND ND 500 400 1,800 13,000 <300 <3,300 -c 2,500 Abbreviation: ND, not done. For the population analyzed. t Measured as the total clonogenic output in LTC after 6 weeks divided by 8 (see Material and Methods). 400 800 600 500 500 500 <600 ? 40 12,000 8,500 20,000 4,500 8,000 13,000 11,000 2 2,200 ND 700 100 300 800 200 400 2 100 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 2981 CD44EXPRESSION ON NORMAL AND CML CELLS 1ll T CFU-GM TOTAL BFU-E LTC-IC CELLS Fig 4. Comparison ofthe distributionof different types of progenitors between theCD34+ CD44++(01and CD34* CD44+++(.l fractions of normal light-density marrow cells with thelight scatter characteristics shown in Fig 1. Values shown are the mean f SEM of measurements of the relative recovery ofprogenitorsfrom each of six experiments calculated in each caseby multiplying the percentages of cells retrieved in each fraction by the corresponding progenitor enrichment observed in that fraction and then normalizingthe data to 100% for the number of Progenitorsdetected in the totalCD34+ population. To determine the nature of the vl0-containing CD44 isoform(s) expressed by 2G1+ cells, RT-PCR was performed. Primers were designed to amplify all vl0-containing CD44 isoforms but to exclude all other sequences lacking this exon. This was achieved by selecting a 5' primer from a region common to all CD44 isoforms and a 3' primer in the last of the alternatively spliced exons (v10). This analysis yielded two major bands of approximately 680 and 480 bp (Fig 3). These correspond to the expected sizes of the CD44 isoforms RI and R2 (672 and 481 bp, respectively), which have been previously demonstrated in various hematopoietic cells.21 Differential expression of CD44 on different normal progenitor populations. In view of the extensive variation in the level of CD44 expression seen on the CD34+ cells present in normal BM, it was of interest to determine whether any lineage-associated changes in CD44 expression might be demonstrable. To examine this possibility, light-density CD34+ cells from six normal BM samples were sorted according to their expression of very high(+ + +) or intermediate levels (+ +) of CD44 and detectable versus undetectable levels of Thy-l. Cells from each of the four subpopulations thus obtained (shown in Fig 1) were then assayed in both clonogenic and LTC-IC assays. The resultant frequencies of the various progenitor types measured are shown in Table 3. The greatest enrichment of BFU-E was obtained in the CD44++Thy-I- fraction and, to a lesser extent, in the CD44++Thy-l+fraction. Veryfew BFU-E were found in the CD44+++fractions (either Thy-l+ or Thy-l -; Fig 4). In contrast, comparing the absolute numbers of colonies recovered, we found that both CFU-GM and LTC-IC were more heterogeneous in their levels of CD44 expression (Table 3 and Fig 4). Most CFU-GM were Thy-I-, and further separa- tion of these cells according to their level of CD44 expression did not result in a selective enrichment of a subpopulation of Thy-l- CFU-GM. Some (8% ? 4%) CFU-GM were also Thy- 1 +.These appeared to be confined primarily to the CD44++ population of CD34+ cells. As found previously, LTC-IC were usually more highly enriched in the Thy-l+ as opposed to the Thy-l- fractions of CD34+ normal BM cells.33However, in terms of total LTC-IC yields, a substantial proportion (68% +- 13%) of all LTC-IC were Thy-l-. Figure 4 shows a comparison of the relative distribution of each of these normal progenitor types between the CD44" and the CD44+++fractions. The difference in the ratio of CD44++ to CD44+++progenitors between CFU-GM (or LTC-IC) and BFU-E was statistically significant ( P < .02 in both cases, Student's t test). Expression of CD44 on CML progenitors is altered. PB samples from five patients with CML were analyzed to determine the level of expression of Thy-l and CD44 on various primitive leukemic cell populations. The frequencies of CFU-GM and BFU-E in each of the four CD34+ subpopulations defined by differences in Thy-l and CD44 expression (gated as indicated in Fig 2) are shown in Table 4. The relative recoveries of these progenitors in the CD44++versus the CD44"' fractions by comparison with normal progenitors are shown in Fig 5. To control for the fact that CML cells might exhibit features unique to circulating and/or activated mobilized progenitors, a series of PB harvests collected after treatment of patients with remission AML or Hodgkin's disease with chemotherapy and administration of G-CSF or GM-CSF and IL-3 were also analyzed. Scatter plots of CD44 versus Thy-l expression by the CD34+ cells in these leukapheresis samples were not noticeably different (data not shown) from those seen for normal BM (Fig 1) or CML PB (Fig 2). Similar CD44 gates were, therefore, used to compare the distribution of different progenitor types amongst the two subpopulations of interest (defined by their levels of CD44 expression) in the CML PB and mobilized normal PB samples. The results of these studies are shown in Table 5 and in Fig 5. The analyses of the CML samples revealed a number of interesting findings. First, as can be seen in Table 4, both Thy-l+ and Thy-l BFU-E and CFU-GM were readily and consistently detected in CML PB. Althoughgenotyping studies of the colonies produced by these sorted progenitors were not performed, it is unlikely from the number originally present in the samples used (Table 1) that any of these sorted progenitors contained a significant proportion of normal cells. Moreover, in a more extensive analysis of CD34+ subpopulations of CML progenitors reported elsewhere,"' the presence of a significant population of Ph+ Thy- 1 CFUGM in CML PB has been directly demonstrated. Second, the number of LTC-IC in the CML PB samples studied was often below the level of detectability. More recent studies4' have shown that cryopreservation selectively kills Ph+ LTCIC, which would explain the unexpectedly low yieldof LTCIC obtained in this study wheremost samples hadbeen frozen before use. Finally, as shown in Fig 5 , the proportion of CFU-GM found in the CD44+++fraction of CD34+ CML ~ + From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 2982 GHAFFARI ET AL Table 4. Progenitor Distributionsin Subpopulations of CD34' CML PB Cells by Their Levels of Expression of CD44 and Thy-l No. of Prooenitors/lOsCellst CD44" Type of Progenitor Evaluated BFU-E Patient No. Thy-l la* 1b* 2 17,000 3 23,000 4 15,000 5 Mean t SEM la* 1b* 2 3 4 5 Mean 2 SEM 5,000 CFU-GM CD44' Thy-l Thy-l' Thy-l + ND 4,800 7,000 5,300 7,300 7,200 6,300 2 500 ND 9,000 13,000 9,900 21,000 12,000 13,000 5 2,100 13,000 3,500 11,000 2,500 14,000 2 2,500 900 2,700 6,700 3,800 6,000 4,100 4,000 t 900 ND 5,300 4,300 3,500 7,900 ND 5,300 2 1,000 ND 7,100 1,800 4 , 7 0 0 2 1,000 11,000 19,000 12,000 24,000 19,000 ND 19,000 2 2,500 18,000 16,000 13,000 20,000 23,000 17,000 2 1,800 700 <500 Patient numbers l a and l b refer to fresh and thawed cells from the same original sample (see Table 1). t For the population analyzed. PB cells was consistently and significantly ( P < .01) elevated by comparison to CFU-GM in normal BM. Interestingly, this was not true for the BFU-E present in CML PB. Moreover, the alteration in CD44 expression by CML CFUGM was not found to be a general feature of normal CFUGM that had been mobilized into the circulation, because the ratio of CD44++ to CD44+++ CFU-GM in leukapheresis harvests was not different from that characteristic of CFUGM in normal BM. DISCUSSION The production of different CD44 isoforms in conjunction with variable degrees of their glycosylation and chondroitin v CML NBM MOB CFU-GM BFU-E CYL MOB NBH CML MOB NBH TOTALCELLS Fig 5. Comparison of the distribution ofBFU-E and CFU-GM in the CD34+ CD44*+ ( 0 )and CD34+ CD44+++(m) fractions of CML PB (CML;n = 6 five patients, one evaluated in duplicate), mobilized blood (MOB; n = 3), and normal bone marrow [NBM; n = 6). Error bars indicate 1 SEM above the mean. sulfate attachment is believed to explain the diversity of adhesion-dependent processes in which CD44 has been implicated."." In this study, we have obtained further evidence that CD44 may be involved in the regulation of early stages of hematopoiesis based on the demonstration of differentiation- and transformation-associated changes in the expression of this gene product on primitive normal andCML cells. Analysis of the CD34+ population in normal BM and CML PB indicated that CD44- cells are not present at a detectable level within this fraction, in contrast with the total light-density cell fraction, of which a small percent (up to approximately 10%)may be identified as CD". Functional studies showed that the LTC-IC in normal marrow express intermediate to very high levels of CD44, thus mirroring the heterogeneous pattern of CD44 expression also exhibited by normal CFU-GM. In contrast, BFU-E in normal BM represent a more homogenous population of cells in terms of their pattern of CD44 expression. It will be interesting to determine whether the subset of CFU-GM expressing very high levels of CD44 are those that are able to bind immobilized hyaluronan4' or that cooperate with the cr4/3, integrin in binding to the C-terminal heparin-binding domain of fibronectin.4' Kansas et all4 also have reported changes in CD44 expression as hematopoietic cells mature, although in their studies, CD44 was seen to be downregulated at later stages of myeloid and erythroid cell maturation. The pattern of CD44 expression on primitive CML cells was found to differ in two respects from that exhibited by normal BM or mobilized PB cells. First, a significantly larger proportion of CML CFU-GM were found to express CD44 at a very high level, and second, a subset of CD34+ cells expressing an exon vl0-containing CD44 isoform(s)that was not detectable in normal BM could be readily detected in all CML PB samples analyzed. Because all normal samples used in this work were frozen, the changes in thepattern of CD44 expres- From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CML CD44 EXPRESSION AND ON NORMAL CELLS 2983 Table 5. Progenitor Distributions in Circulating Subpopulationsof Chemotherapy and Growth Factor Mobilized CD34+ Cells CD44++ Progenitor Evaluated 2,000 BFU-E 4,300 13,000 CFU-GM 1,900 2,100 Sample No. ~ ~ Cells 1 1 2 3 Mean ? SEM 1 2 3 Mean ? SEM 0 ~ Enrichtl CD44"' Recoveryt 86 97 73 6,400 ?100 3,300 6,000 27 3,300 ? 1,300 935 66 5 38 31 2 13 ? 4 73 84 97 62 73 ? 6 4 (%l FWlO' Cells 14 Enricht" Recovaryt (%l 1 5 3 610 290 680 2 260 4,300 16 800 4,100 3,100 5 1,100 9 72 56 4 17 38 39 27 5 29 5 6 Abbreviation: FQ, frequency. * Calculated by dividing the FO/105 sorted cells by the FQ/105 unsorted light-density cells in each individual experiment. t Calculated as described in the legend to Fig 4. sion on CML cells could not be attributed to a general freezethaw artefact. Whether they may contribute to the abnormal adhesive properties previously described for CML cells remains to be established. The mechanism(s) underlying the increased expression of CD44 on CML CFU-GM as well as the increased expression of exon vl0-containing CD44 isoform(s) on more mature CML cells, including some within the CD34+ population, is also unknown. In particular, it is not clear whether these increases are accompanied by concomitant changes in the expression of other CD44 isoforms. Activation is one of multiple mechanisms of CD44 isoform expression described in different cell types.I6 The increase observed here in expression of CD44 on CML CFU-GM may, therefore, be related to their constitutively activated for example, through BCR-ABL stimulation of the ras pathway as a result of the association of p210BCR-ABL with GRB-~/SOS?'~'The product of the BCR-ABL gene has also been found to inhibit p120 GAP which would favor the formation of GTP-bound ras and, hence, its accumulation in an activated form. The results of BCR-ABL transfection experiments have also implicated p210BCR-ABL in the constitutive activation of ras,48 and several studies have indicated that ras activation may alter CD44 expression, by modulating CD44 promotor activity as well as via mechanisms that control CD44 transcript s p l i ~ i n g .It~ is~ interesting , ~ ~ ~ ~ ~to note that modulations of CD44 expression were not observed in cells obtained from patients treated with IL-3 in spite of the described ability of this cytokine to transiently activate the ras pathway." Collectively, these findings suggest multiple mechanisms by which the observed changes in CD44 expression seen at different levels of granulopoiesis in CML could be related to the expression of the BCR-ABL gene in these cells, although they do not explain why such changes should be restricted to the granulopoietic lineage. The identification of a small subset of maturing myeloid cells in normal BM that appear to express different exon vl0-containing isoforms of CD44 is concordant with previous reports demonstrating that a change in CD44 isoform expression is not uniquely associated with malignant transf o r m a t i ~ n * but ~ .may ~~ be more closely correlated with changes in properties that also occur during the development or functional activation of normal The observed increase in expression of exon vl0-containing CD44 isoform(s) on CML cells could simply reflect a selective amplification in CML of a cell type that normally expresses these isoforms. Alternatively, it could reflect a direct effect of malignant transformation on cells that would not normally express exon v10. Support for the latter possibility has been recently suggested by the finding of alterations in CD44 expression on primary cells from patients with other types of (acute) myeloid le~kemia.~' In summary, we have documented the presence of CD44 on LTC-IC in normal human BM and have provided evidence of a heterogeneity in the level of the CD44 expressed on these very primitive cells that is also shared by normal CFU-GM. In contrast, most normal BFU-E express, on average, a lower level of CD44. We have also provided evidence for the expression of different vl0-containing CD44 isoforms in normal human marrow cells. In CML, there is a disproportionate increase in the type of CFU-GM that express very high levels of CD44. These findings, taken together with the abnormal production by CD34' CML cells of exon vl0-containing isoform(s) of CD44, suggest that the control of CD44 mRNA production and processing may be influenced by both normal differentiation mechanisms and those that mediate leukemic transformation. ACKNOWLEDGMENT We thank GayleThomburyandWieslawaDragowskafor op erating the FACS, Giovanna Cameron for the differential analysis, and Irene Edelmann for assistance in manuscript preparation. We and Amgen and Sandoz also thank Dr J. Spinelli for statistical advice for generous gifts of recombinant human growth factors. REFERENCES 1. Verfaillie C, Hurley R, Bhatia R, McCarthy JB: Role of bone marrowmatrix in normal and abnormalhematopoiesis.CritRev OncolHematol 16:201, 1994 2. Berardi AC, Wang A, Levine JD, Lopez P,Scadden DT: Functional isolation and characterization of human hematopoietic stem cells. Science 267:104, 1995 3. Williams DA, Rios M, Stephens C, Pate1 VP Fibronectin and VLA4 in haematopoietic stem cell-microenvironment interactions. Nature 352:438, 1991 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 2984 4. Molineux G , Migdalska A, Szmitkowski M, Zsebo K, Dexter TM: The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor. Blood 78:961, 1991 5. Bodine DM, Seidel NE, Zsebo KM, Orlic D: In vivo administration of stem cell factor to mice increases the absolute number of pluripotent hematopoietic stem cells. Blood 82:445, 1993 6. Papayannopoulou T, Nakamoto B: Peripheralization of hemopoietic progenitors in primates treated with anti-VLA, integrin. Proc Natl Acad Sci USA 90:9374, 1993 7. Raskind WH, Fialkow PJ: The use of cell markers in the study of human hematopoietic neoplasia. Adv Cancer Res 49:127, 1987 8. Eaves CJ, Eaves AC: Cell culture studies in CML. Baillieres Clin Haematol 1:931, 1987 9. Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF: Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 328:342, 1987 10. Gordon MY, Atkinson J, Clarke D, Dowding CR, Goldman JM, Grimsley PG, Siczkowski M, Greaves MF: Deficiency of a phosphatidylinositol-anchored cell adhesion molecule influences haemopoietic progenitor binding to marrow stroma in chronic myeloid leukemia. Leukemia 5:693, 1991 11. Udomsakdi C, Eaves CJ, Swolin B,Reid DS, Barnett MJ, Eaves AC: Rapid decline of chronic myeloid leukemic cells in longterm culture due to a defect at the leukemic stem cell level. Proc Natl Acad Sci U S A 89:6192, 1992 12. Verfaillie CM, McCarthy JB, McGlave PB: Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. J Clin Invest 90:1232, 1992 13. Trowbridge IS, Lesley J, Schulte R, Hyman R, Trotter J: Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues. Immunogenetics 15:299, 1982 14. Kansas GS, Muirhead MJ, Dailey MO: Expression of the C D I K D 1 8 , leukocyte adhesion molecule I, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans. Blood 76:2483, 1990 15. Lewinsohn DM, Nagler A, Ginzton N, Greenberg P, Butcher EC: Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule. Blood 75589, 1990 16. Lesley J, Hyman R, Kincade PW: CD44 and its interaction with extracellular matrix. Adv Immunol 54:271, 1993 17. Underhill C: CD44: The hyaluronan receptor. J Cell Sci 103:293, 1992 18. Toyama-Sorimachi N, Sorimachi H, Tobita Y, Kitamura F, Yagita H, Suzuki K, Miyasaka M: A novel ligand for CD44 is serglycin, a hematopoietic cell lineage-specific proteoglycan. J Biol Chem 270:7437, 1995 19. Screaton GR, Bell MV, Jackson DG, Cornelis F B , Gerth U, Bell JI: Genomic structure of DNAencoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci U S A 89:12160, 1992 20. Mackay CR, Terpe HJ, Stauder R, Martson WL, Stark H, Gunthert U: Expression and modulation of CD44 variant isofoms in humans. J Cell Biol 124:71, 1994 21. Dougherty GJ, Lansdorp PM, Cooper DL, Humphries R K Molecular cloning of CD44R1 and CD44R2, two novel isofoms of the human CD44 lymphocyte вЂњhomingвЂќ receptor expressed by hemopoietic cells. J Exp Med 174:1, 1991 22. Stamenkovic I, Aruffo A, Amiot M, Seed B: The hematopoietic and epithelial forms of CD44 are distinct polypeptides with GHAFFARI ET AL different adhesion potentials for hyaluronate-bearing cells. EMBO J 10:343,1991 23. Horst E, Meijer CJ, Radaskiewicz T, van Dongen JJ, Pieters R, Figdor CG, Hooftman A, Pals ST: Expression of a human homing receptor (CD44) in lymphoid malignancies and related stages of lymphoid development. Leukemia 4:383, 1990 24. Salles G , Zain M, Jiang W, Boussiotis VA, Shipp MA: Alternatively spliced CD44 transcripts in diffuse large-cell lymphomas: Characterization and comparison with normal activated B cells and epithelial malignancies. Blood 82:3539, 1993 25. Koopman G, Heider KH, Horst E, Adolf GR, van den Berg F, Ponta H, Herrlich P, Pals ST: Activated human lymphocytes and aggressive non-HodgkinвЂ™s lymphomas express a homologue of the rat metastasis-associated variant of CD44. J Exp Med 1772397, 1993 26. Hofmann M, Rudy W, Zoller M, Tolg C, Ponta H, Herrlich P, Gunthert U: CD44 splice variants confer metastatic behavior in rats: Homologous sequences are expressed in humantumor cell lines. Cancer Res 51:5292, 1991 27. Matsumura Y, Tarin D: Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet 340: 1053, 1992 28. Penno MB, August JT, Baylin SB, MabryM, Linnoila RI, Lee VS, Croteau D, Ling Yang X, Rosada C: Expression of CD44 in human lung tumors. Cancer Res 54:1381, 1994 29. Finn L, Dougherty G , Finley G, Meisler A, Becich M, Cooper DL: Alternative splicing of CD44 pre-mRNA in human colorectal tumors. Biochem Biophys Res Commun 200:1015, 1994 30. Miyake K, Medina KL, Hayashi SI, Ono S, Hamaoka T, Kincade PW: Monoclonal antibodies to Pgp-lKD44 block lymphohemopoiesis in long-term bone marrowcultures. J Exp Med 171:477, 1990 31. Gunji Y, Nakamure M, Hagiwara T, Hayakawa K, Matsushita H, Osawa H, Nakayoshi K, Nakauchi H, Yanagisawa M, Muira Y, Suda T Expression and function of adhesion molecules on human hematopoietic stem cells: CD34вЂ™ LFA-1- cells are more primitive than CD34вЂ™ LFA-lвЂ™ cells. Blood 80:1195, 1992 32. Schmitt C, Eaves CJ, Lansdorp PM: Expression of CD34 on human B cell precursors. Clin Exp Immunol 85:168, 1991 33. Craig W, Kay R, Cutler RL, Lansdorp PM: Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med 177:1331, 1993 34. Dougherty GJ, Cooper DL, Memory JF, Chiu RK: Ligand binding specificity of alternatively-spliced CD44 isoforms. Recognition of hyaluronan by CD44R1. J Biol Chem 269:9074, 1994 35. Droll A, Dougherty ST, Chiu RK, Dirks JF, McBride WH, Cooper DL, Dougherty GJ: Adhesive interactions between alternatively spliced CD44 isoforms. J Biol Chem 270:11567, 1995 36. Thomas TE, Abraham SJR, Otter A J , Blackmore EW, Lansdorp PM: High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens. J Immunol Methods 154:245, 1992 37. Cashman J, Eaves AC, Eaves CJ: Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 38. Sutherland W, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ: Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci U S A 87:3584, 1990 39. Sauvageau G , Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, Reid DS, Largman C, Lawrence HJ, Humphries RK: Differential expression of homeobox genes in functionally distinct CD34вЂ™ subpopulations of human bone marrow cells. Proc Natl Acad Sci U S A 91:12223, 1994 40. Petzer A, Ponchio L, Ghaffari S, Cashman J, Lansdorp P, Eaves A, Eaves C: Mobilization of normal LTC-IC in the blood of From www.bloodjournal.org by guest on December 22, 2014. For personal use only. CD44 EXPRESSION ON NORMAL AND CML CELLS untreatedCMLpatientswithhigh WBCcounts.Blood MWa, 1994 (abstr, suppl 1) 41. Smadja-Joffe F, Legras S,Li Y,Clay D, Robin E, Morimoto K, LeBousse-Kerdiles MC, Jasmin C: Hematopoietic progenitors from normal human bone marrow adhere tohyalmnan (HA). This adhesion is mediated by CD44 and involves preferentially granulomacrophagic progenitors. Blood M416a. 1994 (abstr, suppl 1) 42. Verfaillie CM, Benis A, Lida J, McGlave PB, McCarthyJB: Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin:Cooperationbetweentheintegrin a4P1 and the CD44 adhesion receptor. Blood 84:1802, 1994 43. Camp RL, Kraus TA, Birkeland ML, Pure E High levels of CD44 expression distinguish virgin from antigen-primed B cells. J Exp Med 173:763, 1991 44. Bodine DM, Seidel N E , GaleMS,NienhuisAW,OrlicD: Efficient retrovirus transductionof mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood bytreatmentwith granulocyte colony-stimulating factor and stem cell factor. Blood 84:1482, 1994 45. Pendergast AM, Quilliam LA, Cripe LD, Bassing CH, Dai Z, Li N, Batzer A, Rabun KM, Der U, Schlessinger J, Gishizky ML: BCR-ABL-induced oncogenesis is mediated by direct interaction with theSH2 domainof the GRB-2 adaptor protein. Cell 75:175, 1993 46. Puil L, Liu J, Gish G, Mbamalu G, Bowtell D, Pelicci PG, ArlinghausR,Pawson T Bcr-Ab1 oncoproteins binddirectlyto activators of the ras signalling pathway. EMBO J 13:764, 1994 47. Tauchi T, Boswell HS, Leibowitz D, Broxmeyer HE: Coupling between p2lObcr-ab1 and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to Ras activation pathway. J Exp Med 179:167, 1994 2985 48.Skorski T, Kanakaraj P, KuDH,Nieborowska-SkorskaM, Canaani E, Zon G, Perussia B, CalabrettaB: Negative regulation of p12OGAP GTPase pmoting activity by p2lObcr/abl: Implication for rasdependent Philadelphia chromosome positivecell growth. J Exp Med 179:1855, 1994 49. Hofmann M, Rudy W, Gunthert U, Zimmer SG, Zawadzki V, Zoller M, Lichtner RB, Hemlich P, Ponta H: A link between ras and metastatic behaviorof tumor cells: ras induces CD44 promotor activity and leadsto low-level expressionof metastatic-specific variants of CD44 in CREF cells. Cancer Res 53:1516, 1993 50. Jamal HH, Cano-Gauci DF, Buick RN, Filmus J: Activated ras and src induce CD44 overexpression in rat intestinal epithelial cells. Oncogene 9:417, 1994 51. Satoh T, Nakafuku M. Miyajima A, Kaziro Y Involvement of ras p21 protein in signal-transduction pathways from interleukin 2,interleukin 3, andgranulocytdmacrophagecolony-stimulating factor, but not from interleukin4. Proc Natl AcadSci U S A 88:3314, 1991 52. Arch R, Wirth K, Hofmann M, Ponta H, Matzku S,Herrlich P, Zoller M: Participation innormal immune responses ofa metastasisinducing splice variant of CD44. Science 257:682, 1992 53. Bennett KL, Jackson DG, Simon JC, Tanczos E, Peach R, Modrell B, Stamenkovic I, Plowman G, Aruffo A: CD44 isofom containing exon V3 are responsible for the presentation of heparinbinding growth factor. J Cell Biol 128:687, 1995 54. Jackson DG, Bell JI, Dickinson R, TimansJ, Shields J, Whittle N: Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon. J Cell Biol 128:673, 1995 55. Ghaffari S, Dougherty G, Lansdorp P, Eaves C: Abnormal patterns of CD44 expression on primitive cells in CML and AML. Blood 84:154a, 1994 (abstr, suppl 1) From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 1995 86: 2976-2985 Differentiation-associated changes in CD44 isoform expression during normal hematopoiesis and their alteration in chronic myeloid leukemia S Ghaffari, GJ Dougherty, PM Lansdorp, AC Eaves and CJ Eaves Updated information and services can be found at: http://www.bloodjournal.org/content/86/8/2976.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
© Copyright 2021 Paperzz